Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Abstract

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock "Pd1" (Prunus tomentosa) on sweet cherry 'Shuguang2' scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock-scion interactions.

Keywords: DNA methylation; Prunus tomentosa; rootstock–scion interactions; transcriptome; whole-genome bisulfite sequencing.

Figure 1

Growth and anatomical observation of shoot tips. (A) Grafting plants at the growth stagnation stage (195 days after grafting). Red circles mark shoot tips. (B) Anatomical observation of shoot tips. SG-Pd1: “Shuguang2” sweet cherry grafted onto Pd1 rootstock (dwarf); SG-WT: “Shuguang2” sweet cherry grafted onto wild-type rootstock (vigorous).

Figure 2

The effect of rootstock on scion growth. (A) The growth of “Shuguang2” grafted buds. (B) New branch growth of 4-year-old grafted plants. Significant differences from the fifteen biological replicates were calculated using t-test and indicated by * (p value < 0.05). SG-Pd1: “Shuguang2” sweet cherry grafted onto Pd1 rootstock (dwarf); SG-WT: “Shuguang2” sweet cherry grafted onto wild-type rootstock (vigorous).

Figure 3

The content of GAs and IAA in shoot tips. ND indicates “not detected”. Significant differences were evaluated using t-test and indicated by ** (p value < 0.01), *** (p value < 0.001), and **** (p value < 0.0001). SG-Pd1: “Shuguang2” grafted onto Pd1 rootstock (dwarf); SG-WT: “Shuguang2” grafted onto wild-type rootstock (vigorous).

Figure 4

Relative expression analysis of genes encoding DNA methylation-related enzymes using RT-qPCR. (A) Genes encoding de novo methylation-related enzymes. (B) Genes encoding demethylation-related enzymes. (C) Genes encoding methylation maintenance-related enzymes. Significant differences were assessed by t-test and are indicated by * (p value < 0.05), ** (p value < 0.01), and *** (p value < 0.001), with ns indicating “not significant”.

Figure 5

Features of the sweet cherry scion methylome. (A) The methylation level of sweet cherry scions. (B) Relative proportions of mCpG, mCHG, and mCHH in total mC. (C) DNA methylation of chromosomes in the sweet cherry genome. (D) The methylation level of each chromosome in sweet cherry. (E) Methylation density. (F) The DNA methylation features of genes and TEs. Mds_S and Mds_L indicated shoot tips and leaves from SG-Pd1, respectively; Mwt_S and Mwt_L indicated shoot tips and leaves from SG-WT, respectively.

Figure 6

Dynamic changes in DNA methylation after grafting. (A) The DNA methylation level of each sample. (B) DNA methylation characteristics of genes and TEs. (C) Principal component analysis (PCA) of total mC, mCpG, mCHG, and mCHH. Mds_S and Mds_L indicate shoot tips and leaves from SG-Pd1, respectively; Mwt_S and Mwt_L indicate shoot tips and leaves from SG-WT, respectively.

Figure 7

Identification of DMRs. (A) The number of DMRs identified in Mds_S vs. Mwt_S (shoot tips from SG-Pd1 vs. shoot tips from SG-WT) and Mds_L vs. Mwt_L (leaves from SG-Pd1 vs. leaves from SG-WT). (B) The proportion of CpG-DMRs, CHG-DMRs, and CHH-DMRs. (C) The proportion of hyper-DMRs and hypo-DMRs. (D) The number of DMRs on chromosomes (chr1–chr8). (E) Boxplot of DNA methylation changes in DMRs.

Figure 8

Differentially methylated gene (DMG) analysis. (A) The number of DMGs identified in Mds_S vs. Mwt_S (shoot tips from SG-Pd1 vs. shoot tips from SG-WT) and Mds_L vs. Mwt_L (leaves from SG-Pd1 vs. leaves from SG-WT). (B) Venn diagram analysis of DMGs in different sequence contexts (mCpG-DMGs, mCHG-DMGs, and mCHH-DMGs) and regions (promoter, exon, and intronic and intergenic regions). (C) Venn diagram analysis of DMGs. (D) KEGG pathway enrichment analysis of DMGs.

Figure 9

Differentially expressed gene (DEGs) analysis. (A) The number of DEGs. (B) Venn diagram of DEGs in shoot tips and leaves. (C) Volcano plot showing DEGs between shoot tips and leaves. (D) KEGG enrichment analysis was separately conducted on the DEGs in the shoot tips and leaves. WT_S and DS_S indicate shoot tips of SG-WT and SG-Pd1, respectively; WT_L and DS_L indicate leaves of SG-WT and SG-Pd1, respectively.

Figure 10

Correlation analysis between DNA methylation and gene expression. (A) Venn diagram analysis of DEGs and DMGs in shoot tips and leaves. (B) Venn diagram analysis of overlapping genes in shoot tips and leaves. (C) Distributions of methylation levels within genes partitioned by different expression levels: rank1 is the lowest, and rank6 is the highest. (D) Differential expression levels of all genes, hypermethylated genes, and hypomethylated genes, displayed as boxplots. Wilcoxon p values indicated by ** (p value < 0.005). In addition, ns indicates “not significant”.